Transcriptome-based identification and characterization of genes responding to imidacloprid in Myzus persicae

Myzus persicae is a serious and widespread agricultural pest, against which, imidacloprid remains an effective control measure. However, recent reports indicate that this aphid has evolved and developed resistance to imidacloprid. This study aimed to elucidate the underlying mechanisms and genetic basis of this resistance by conducting comparative transcriptomics studies on both imidacloprid-resistant (IR) and imidacloprid-susceptible (IS) M. persicae. The comparative analysis identified 252 differentially expressed genes (DEGs) among the IR and IS M. persicae transcriptomes. These candidate genes included 160 and 92 genes that were down- and up-regulated, respectively, in the imidacloprid-resistant strain. Using functional classification in the GO and KEGG databases, 187 DEGs were assigned to 303 functional subcategories and 100 DEGs were classified into 45 pathway groups. Moreover, several genes were associated with known insecticide targets, cuticle, metabolic processes, and oxidative phosphorylation. Quantitative real-time PCR of 10 DEGs confirmed the trends observed in the RNA sequencing expression profiles. These findings provide a valuable basis for further investigation into the complicated mechanisms of imidacloprid resistance in M. persicae.

In addition, a number of studies have associated high expression levels of cytochrome P450 genes with neonicotinoids resistance in insects 4 . For example, the overexpression of CYP6CY3, CYP6G1, CYP6CM1 and CYP4C64 has been associated with imidacloprid resistance in M. persicae, Drosophila melanogaster and Bemisia tabaci 10,13,14 . An increase in the activities of detoxification enzymes, such as glutathione S-transferases (GSTs) and carboxylesterases, is also known to be associated with imidacloprid resistance in aphids 15 . Although reduced target-site sensitivity and enhanced metabolic detoxification are known to contribute to imidacloprid resistance, it is also possible that both resistance mechanisms and adaptation strategies are complex processes that involve an array of metabolic and genetic factors and that such complex processes are responsible for the development of imidacloprid resistance in M. persicae.
Global surveys of transcriptional changes in insecticide-treated insects could help elucidate the metabolic and regulatory mechanisms that underlie insecticide resistance. Currently, powerful next-generation RNA sequencing (RNA-Seq) technology can be used to determine the gene expression profiles and, thereby, helping to elucidate the development of insecticide resistance [16][17][18] . In this study, high-throughput RNA-Seq was used to determine the transcriptome profiles of imidacloprid-resistant (IR) and imidacloprid-susceptible (IS) M. persicae, with a focus on genes that could provide insight into the mechanisms of physiological adaptation of insects to imidacloprid stress.  Table S2). Most DEGs in the BP category were putatively attributed to "signal transduction", "single organism signaling", "cell communication", and "signaling", those in the CC category were attributed to "extracellular region" and "cytoskeleton" and those in the MF category were attributed to "structural molecule activity", "metal ion binding", "cation binding", and "structural constituent of cuticle".

RNA
Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that 100 DEGs were annotated for M. persicae. These annotated genes were classified into 45 groups based on the secondary pathway hierarchy (Supplementary Table S3). The major pathways included metabolic pathways (21 proteins), oxidative phosphorylation (9 proteins), Hippo signaling pathway -fly (5 proteins), and phenylalanine metabolism (4 proteins; Fig. 3). In the database of the present study, phenylalanine metabolism (api00360), oxidative phosphorylation (api00190), Hippo signaling pathway -fly (api04391), tyrosine metabolism (api00350), and ECM-receptor interaction (api04512) pathways were found to be with higher corrected P-values < 0.05. These annotations establish an invaluable basis for elucidating the specific processes, functions, and pathways involved in the imidacloprid resistance of M. persicae.
qRT-PCR validation of DEGs. qRT-PCR analysis of 10 randomly selected genes confirmed the expression trends observed in the RNA-Seq results (Fig. 4, Supplementary Table S4), thereby, suggesting that the DEG profiles were reliable. Increases in the expression of the selected genes were small, and the biological relevance of these changes is likely minimal. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The green peach aphid, M. persicae, is an economically important pest that is typically controlled using insecticides. However, the irrational use of imidacloprid has promoted the rapid development of insecticide resistance  www.nature.com/scientificreports www.nature.com/scientificreports/ in many M. persicae populations, resulting in the failure in controlling the pest [6][7][8] . Undoubtedly, the development of insecticide resistance is complex and is a common adaptation to insecticide exposure. This study aimed to identify imidacloprid-responsive genes in M. persicae, to establish a basis for investigating the responses and adaptive physiological changes that contribute to insect resistance.
For insect pests, insecticide exposure can be considered a common environmental stress and the cuticle is the first and main barrier for insects against environmental stresses. As such, cuticular proteins have been reported to play crucial roles in the insecticide resistance and tolerance of a variety of insect species, including M. persicae, Lymantria dispar, Aphis gossypii and Plutella xylostella 13,[16][17][18]    www.nature.com/scientificreports www.nature.com/scientificreports/ The ABC transporters are responsible for the translocation of a variety of substrates (e.g. nutrients, lipids, inorganic ions and xenobiotics) and can be categorised into eight subfamilies from ABCA to ABCH 19 . The ABC transporters present in the blood-brain barrier of insects can protect the nervous system from insecticides 20 and are reportedly involved in insecticide resistance 21,22 . Indeed, the contribution of ABC transporters to insecticide resistance has been reported for 27 different insecticides including, imidacloprid, pyrethroids, and avermectin 21,23,24 . With regard to imidacloprid, ABC transporters have been reported to enhance its exclusion from the brain and to hinder its access to target sites 24 . Recently, the down-regulation of these genes was reported to be linked to insecticide resistance 22,25 . Similarly, in our study, it was found that ABCG23 (ABCG23, 111040927) was down-regulated in IR M. persicae, which suggests that the ABC transporter is involved in imidacloprid metabolism and transport.
Glutathione S-transferases (GSTs) are a widespread superfamily of genes that occur in almost all living organisms and participate in a variety of cellular physiological processes, including the detoxification of harmful endobiotic and xenobiotic compounds. Insect GSTs are generally categorised into two main groups: cytosolic and microsomal, based on their cellular location. In insects, most GSTs are cytosolic proteins and are classified as delta, epsilon, omega, sigma, theta, and zeta 26 . In recent years, a number of studies have investigated the correlation between insect GST genes and insecticide resistance. These studies have demonstrated that insect GSTs play important roles in insecticide detoxification and eliminate the oxidative stress caused by insecticide exposure 27 . The up-regulation of GST genes has been associated with insecticide detoxification 28 . However, the down-regulation of GST genes following insecticide exposure has also been reported in several insect species. In Leptinotarsa decemlineata, for example, the expression of LdGSTe4 and LdGSTe6 was significantly down-regulated after cyhalothrin, fipronil, and endosulfan exposure, and that of PxGSTd2, PxGSTe2, PxGSTe5, PxGSTo1, PxGSTs1, PxGSTs2, and PxGSTt1 was down-regulated by β-cypermethrin exposure 29 . Similarly, our results indicated that GST (111036096, 111036826) was down-regulated in IR M. persicae. Bautista et al. suggested that this phenomenon may be an adaptive mechanism to insecticide pressure and an energy trade-off strategy to ensure that energy is allocated to the most effective genes responsible for detoxification when stimulated by insecticide exposure 30 . This suggests that GST plays a relatively minor role in the imidacloprid metabolism of M. persicae. However, because insect GST genes exhibit a variety of expression responses to insecticide exposure 29 , functional studies are needed to examine this hypothesis and identify the specific GST members involved in insecticide detoxification.
Trypsin is a serine protease responsible for digestion; it also contributes to insecticide detoxification 31 . Recently, Zhu et al. (2015) reported that the midgut trypsin activity of Bt-resistant Spodoptera frugiperda was relatively lower than that of a susceptible strain 32 . Indeed, in our study, trypsin gene (111033016) was also down-regulated in IR M. persicae. These findings suggest that the expression and function of trypsin are associated with insecticide resistance. However, little is known about the exact role of trypsin in imidacloprid resistance. It has been demonstrated that the lack of midgut trypsin made some insects adapt to insecticide toxins by a mechanism where incomplete or non-activation of the protoxin occurs, and finally induced resistance development 33 .
Mitochondria play critical roles in a variety of cellular processes, among which energy generation is the most critical and which is primarily achieved through coupled oxidative phosphorylation. The electron transport chain (ETC) consists of four macromolecular protein complexes (complex I-IV), that coordinate to maintain mitochondrial inner membrane potential 34 . In this study, the mitochondrial complex-related genes NADH dehydrogenase (ETC I, 111029398), succinate dehydrogenase (ETC II, 111027244), cytochrome b-c1 complex (ETC III, 111038823, 111027246), and cytochrome c oxidase (ETC IV, 111034852) were all down-regulated in IR M. persicae, which indicated that imidacloprid exposure reduced the expression of ETC I, II, III, and IV component genes. It is possible that the respiration and energy production of IR aphids may have been weakened by imidacloprid exposure. There is no evidence that the ETC contributed to the enhanced imidacloprid tolerance, but the apparent alteration in the expression of complex I-IV in IR M. persicae clearly associates the mitochondrial complex-related genes with imidacloprid resistance. Previous studies have suggested that the overexpression of cytochrome P450s is the primary reason for neonicotinoid resistance 35,36 . Indeed, CYP6CY3, which is a cytochrome P450 gene in M. persicae, has been suggested to play a primary role in the development of insecticide resistance 4,13 . However, the association between mitochondrial complexes and cytochrome P450s with the resistance of M. persicae to imidacloprid requires further investigation.
In conclusion, this study provides, to our knowledge, the first description of genes related to imidacloprid resistance in M. persicae using an RNA-Seq approach. The results indicate that the response patterns of aphids are complex during the development of imidacloprid resistance, as demonstrated by changes in the expression of genes involved cuticle structure, binding, metabolic processes, and oxidative phosphorylation. Further investigations are needed to assess the specific roles of these genes in the response of M. persicae to the stress of insecticide exposure. These findings will provide a basis for investigating the development and mechanisms of insecticide resistance.

Materials and Methods
Aphid strains. One IS strain and one IR strain of M. persicae were used. The IS strain was obtained from tobacco in Guizhou province, China, in 2009, and was subsequently reared in the absence of insecticides. The IR strain was generated from the IS population under continuous imidacloprid selection pressure 37 . In this study, the imidacloprid resistance of the IR strain was ~45-fold greater than that of the IS strain. Both the IS and IR strains were maintained on tobacco plants at 23 Table 2. Primers used for qRT-PCR validation of differentially expressed genes.